Evolutionary conservation of structure-function relationships in proteins allows us to integrate information from various species and make inferences about biological phenomena in different organisms. Over the next decade a basic understanding of the function of most genes will be obtained for several model organisms. Dictyostelium is a social amoeba whose study has contributed to our understanding of diverse biological processes common to many eukaryotes. The in-depth study of model organisms such as Dictyostelium will inform us about human biology and this will inevitably aid in the detection and treatment of disease. This proposal describes a continuing effort to sequencing about one-half of the Dictyostelium genome. The previous project period focused on feasibility studies and the sequencing of chromosome 6. This proposal targets chromosomes 4 and 5 which together constitute about one-third of the 34-Mb genome. We will use whole chromosome shotgun sequencing combined with sample sequencing of a minimum overlapping set of large-insert clones to generate contiguous and highly accurate sequence. The assembled DNA will be integrated with a high-resolution physical map from which finishing experiments will be planned and carried out to complete the sequence. The sequence will be richly annotated and submitted to public databases. All raw sequence data will be made available as soon as possible via our website. The availability of complete genome sequences promises to accelerate research into fundamental mechanisms of cell and developmental biology and the challenge now is to define the genes contained in those genomes and determine their function. Microorganisms that are amenable to high-throughput methods of investigation such as Dictyostelium have been and will continue to be instrumental in this endeavor. The Dictyostelium genome sequence will provide the first detailed glimpse of a biological system that is more complex than single-celled organisms, yet still simple enough to understand completely in the foreseeable future. The genomic data will allow specialists in all areas of biology to enrich the initial findings and make useful extrapolations to other species. Complete sequence data obtained from experimentally tractable organisms, such as Dictyostelium, will continue to stimulate genomic investigations in many systems, including humans.